PROCESSES FOR PURIFICATION OF PROTEINS
The invention is directed to a method for purifying a protein. The method involves providing a sample containing the protein, processing the sample through a capture chromatography resin, inactivating viruses in the sample, and processing through at least one depth filter and ion-exchange membrane.
1 . A method for purifying a protein comprising:
a. providing a sample containing the protein;
b. processing the sample through a capture chromatography resin to provide a first eluate comprising the protein;
c. inactivating viruses in the first eluate to provide an inactivated eluate comprising the protein;
d. processing the inactivated eluate through at least one depth filter to provide a filtered eluate comprising the protein; and
e. processing the filtered eluate through at least one ion-exchange membrane to provide a second eluate comprising the protein.
2 . The method of claim 1 wherein the depth filtration step and the ion-exchange membrane step are provided in a filter train.
3 . The method of claim 1 wherein the capture chromatography resin is selected from the group consisting of an affinity resin, an ion exchange resin, mixed-mode resin, and a hydrophobic interaction resin.
4 . The method of claim 1 wherein the capture chromatography resin is selected from the group consisting of a protein A resin, a protein G resin, a protein A/G resin, and a protein L resin.
5 . The method of claim 1 wherein the protein is selected from the group consisting of a protein fragment, an antibody, a monoclonal antibody, an immunoglobulin, and a fusion protein.
6 . The method of claim 1 wherein the sample is a cell culture.
7 . The method of claim 1 wherein the sample is clarified prior to processing through the capture chromatography resin.
8 . The method of claim 7 wherein the sample is clarified by a clarification method selected from the group consisting of centrifugation, microfiltration, ultrafiltration, depth filtration, sterile filtration, and treatment with a detergent.
9 . The method of claim 1 wherein the viral inactivation comprises a method selected from the group consisting of treatment with acid, detergent, chemicals, nucleic acid cross-linking agents, ultraviolet light, gamma radiation, and heat.
10 . The method of claim 9 wherein viral inactivation comprises lowering the pH of the first eluate to a pH of from about 3 to about 4.
11 . The method of claim 10 wherein the first eluate is incubated for about 30 to about 90 minutes during viral inactivation.
12 . The method of claim 1 wherein the inactivated eluate is adjusted to pH 5 to 10 before depth filtration step.
13 . The method of claim 1 wherein the depth filtration step comprises filtration through at least one depth filter.
14 . The method of claim 1 wherein the depth filtration step comprises filtration through at least two depth filters arranged in series or in parallel.
15 . The method of claim 1 wherein the depth filtration step is followed by a capsule sterile filtration step.
16 . The method of claim 1 wherein the ion-exchange membrane comprises a Q membrane.
17 . The method of claim 16 wherein the Q membrane step is conducted in flow-through mode.
18 . The method of claim 1 wherein the ion-exchange membrane step is followed by a capsule sterile filtration step.
19 . The method of claim 1 wherein the inactivated eluate is processed through one depth filter and the filtered eluate is processed through the ion-exchange membrane in series.
20 . The method of claim 1 wherein the second eluate is further subjected to an additional chromatography step.
21 . The method of claim 20 wherein the additional chromatography step is selected from the group consisting of hydrophobic interaction chromatography, mixed mode chromatography, and cation exchange chromatography.
22 . The method of claim 1 wherein the second eluate is further subjected to a nanofiltration step.
23 . The method of claim 1 wherein the second eluate is further subjected to an ultrafiltration and diafiltration step.
24 . A method for purifying a protein comprising:
a. providing a sample containing the protein;
b. clarifying the sample to provide a clarified sample;
c. processing the clarified sample through a capture chromatography resin to provide a first eluate comprising the protein;
d. inactivating viruses in the first eluate to provide an inactivated eluate comprising the protein;
e. processing the inactivated eluate through at least one depth filter to provide a filtered eluate comprising the protein;
f. processing the filtered eluate through at least one ion-exchange membrane to provide a second eluate comprising the protein;
g. processing the second eluate through an additional chromatography resin to provide a third eluate comprising the protein;
h. subjecting the third eluate to nanofiltration to provide a nanofiltered eluate comprising the protein; and
i. subjecting the nanofiltered eluate to ultrafiltration and diafiltration.
25 . The method of claim 24 wherein the additional chromatography resin comprises a mixed-mode chromatography resin.
26 . The method of claim 25 , wherein the processing of the second eluate through the additional mixed-mode chromatography resin comprises one or more chromatography techniques selected from the group consisting of anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.
27 . The method of claim 26 wherein the processing of the second eluate through the additional mixed-mode chromatography resin comprises a combination of anion exchange and hydrophobic interaction chromatography mechanisms.
28 . The method of claim 26 wherein the mixed-mode chromatography column can be operated in flow-through or bind-elute mode.
29 . The method of claim 24 wherein the additional chromatography resin comprises a cation exchange resin.
30 . The method of claim 29 , wherein the processing of the second eluate through the additional mixed-mode chromatography resin comprises one or more chromatography techniques selected from the group consisting of anion exchange, cation exchange, hydrophobic interaction, hydrophilic interaction, hydrogen bonding, pi-pi bonding, and metal affinity.
31 . The method of claim 30 wherein the processing of the second eluate through the additional mixed-mode chromatography resin comprises a combination of anion exchange and hydrophobic interaction chromatography mechanisms.
32 . The method of claim 29 wherein the cation exchange chromatography column is operated in bind-elute mode.